Medical products comprising a haemocompatible coating, production and use thereof

ABSTRACT

The invention relates to the use of polysaccharides, that comprise the sugar building unit N-acylglucosamine, for the preparation of hemocompatible surfaces as well as methods for the hemocompatible coating of surfaces with these polysaccharides, which are classified to be the common biosynthetic precursor substances of heparin and heparansulphates. Described are medical devices coated according to invention, especially stents, which comprise paclitaxel as antiproliferative active agent as well as the use of these stents for the prevention of restenosis.

[0001] The invention concerns the utilisation of polysaccharidescontaining the sugar building block N-acylglucosamine for thepreparation of hemocompatible surfaces of medical devices, methods forthe hemocompatible coating of surfaces with said polysaccharides as wellas medical devices with these hemocompatible surfaces.

[0002] In the human body the blood gets only in cases of injuries incontact with surfaces other than the inside of natural blood vessels.Consequently the blood coagulation system gets always activated toreduce the bleeding and to prevent a life-threatening loss of blood, ifblood gets in contact with foreign surfaces. Due to the fact that animplant also represents a foreign surface, all patients, who receive animplant, which is in permanent contact with blood, are treated for theduration of the blood contact with drugs, so called anticoagulants, thatsuppress the blood coagulation, so that considerable side effects haveto be taken in account.

[0003] Whilst the usage of vessel supports, so-called stents, thedescribed risk of thrombosis also occurs as one of the risk factors inblood bearing vessels. In cases of vessel strictures and sealings due toe.g. arteriosclerotic changes especially of the coronary arteries thestent is used for the expansion of the vessel walls. It fixes limefragments in the vessels and improves the flow properties of the bloodinside the vessel as it smoothens the surface of the interior space ofthe vessel. Additionally a stent leads to a resistance against elasticrestoring forces of the expanded vessel part. The utilised material ismostly medicinal stainless steel.

[0004] The stent thrombosis occurs in less than one percent of the casesalready in the cardio catheter laboratory as early thrombosis or in twoto five percent of the cases during the hospital recreation. In aboutfive percent of the cases vessel injuries due to the intervention arecaused because of the arterial lock and the possibility of causingpseudo-aneurysms by the expansion of vessels exists, too. Additionallythe continuous application of heparin as anticoagulant increases therisk of bleeding.

[0005] An additional and very often occuring complication is restenosis,the resealing of the vessel. Although stents minimise the risk of arenewed sealing of the vessel they are until now not totally capable ofhindering the restenosis. The rate of resealing (restenosis) afterimplantation of a stent is with up to 30% one of the main reasons of arepeated hospital visit for the patients.

[0006] An exact conceptual description of the restenosis does not existin the professional literature. The mostly used morphologic definitionof the restenosis is that after a successful PTA (percutaneoustransluminal angioplasty) the restenosis is defined as a reduction ofthe vessel diameter to less than 50% of the normal one. This is anempirically defined value of which the hemodynamic relevance and itsrelation to clinical symptomatics lacks of a massive scientific basis.In praxis the clinical aggravation of the patient is often viewed as asign for a restenosis of the formerly treated vessel part.

[0007] The vessel injuries caused during the implantation of the stentsarise inflammation reactions, which play an important role for thehealing process during the first seven days. The herein concurrentprocesses are among others connected with the release of growth factors,which initiate an increased proliferation of the smooth muscle cells andlead with this to a rapid restenosis, a renewed sealing of the vesselbecause of uncontrolled growth. Even after a couple of weeks, when thestent is grown into the tissue of the blood vessel and totallysurrounded by smooth muscle cells, cicatrisations can be too distinctive(neointima hyperplasia) and lead to not only a coverage of the stentsurface but to the sealing of the total interior space of the stent.

[0008] It was tried vainly to solve the problem of restenosis by thecoating of the stents with heparin (J. Whörle et al., European HeartJournal (2001) 22, 1808-1816). Heparin addresses as anticoagulant onlythe first mentioned cause and is moreover able to unfold its totaleffect only in solution. This first problem is meanwhile almost totallyavoidable medicamentously by application of anticoagulants. The furtherproblem is intended to be solved now by inhibiting the growth of thesmooth muscle cells locally on the stent. This is carried out by e.g.radioactive stents or stents, which contain pharmaceutical activeagents.

[0009] Consequently there is a demand on non-thrombogeneous,hemocompatible materials, which are not detected as foreign surface andin case of blood contact does not activate the coagulation system andlead to the coagulation of the blood, with which an important factor forthe restenosis stimulating processes is eliminated. Support is supposedto be guaranteed by addition of active agents which shall suppress theinflammation reactions or which shall control the healing processaccompanying cell division.

[0010] The undertakings are enormous on this area of producing a stentwhich can reduce the restenosis in this manner or eliminate totally.Herein different possibilities of realisation are examined in numerousstudies. The most common construction type consists of a stent, which iscoated with a suitable matrix, usually a biostable polymer. The matrixincludes an antiproliferative or antiphlogistic agent, which is releasedin temporally controlled steps and shall suppress the inflammationreactions and the excessive cell division.

[0011] U.S. Pat. No. 5,891,108 reveals for example a hollow mouldedstent, which can contain pharmaceutical active agents in its interior,that can be released throughout a various number of outlets in thestent. Whereas EP-A-1 127 582 describes a stent that shows on itssurface ditches of 0.1-1 mm depth and 7-15 mm length, which are suitablefor the implementation of an active agent. These active agent reservoirsrelease, similarly to the outlets in the hollow stent, the containedpharmaceutical active agent in a punctually high concentration and overa relatively long period of time, which leads to the fact, that thesmooth muscle cells are not anymore or only very delayed capable ofenclosing the stent. As a consequence the stent is much longer exposedto the blood, what leads again to increased vessel sealings bythrombosis (Liistro F., Colombo A., Late acute thrombosis afterpaclitaxel eluting stent implantation. Heart (2001) 86 262-4).

[0012] One approach to this problem is represented by thephosphorylcholine coating of Biocompatibles (WO 0101957), as herephosphorylcholine, a component of the erythrocytic cell membrane, shallcreate a non thrombogeneous surface as ingredient of the coated nonbiodegredable polymer layer on the stent. Dependent of its molecularweight the active agent is absorbed by the polymer containingphosphorylcholine layer or adsorbed on the surface.

[0013] Object of the present invention is to provide hemocompatiblycoated medical devices as well as methods of hemocompatible coating andthe use of hemocompatibly coated medical devices, especially stents, toprevent or reduce undesired reactions as for example restenosis.

[0014] Especially object of the present invention is to provide stentswhich permit a continuous controlled ingrowth of the stent—on the oneside by suppression of the cellular reactions in the primal days andweeks after implantation by the support of the selected agents and agentcombinations and on the other side by providing an athrombogeneous resp.inert resp. biocompatible surface, which guarantees that with thedecrease of the agent's influence no reactions to the existing foreignsurface take place which also can lead to complications in a long term.

[0015] The intentions of creating a nearly perfect simulation of thenative athrombogeneous conditions of that part of a blood vessel that isallocated on the blood side are enormous. EP-B-0 333 730 describes aprocess to produce hemocompatible substrates by recess, adhesion and/ormodification and anchorage of non thrombogeneous endothelic cell surfacepolysaccharide (HS I). The immobilisation of this specific endotheliccell surface proteoheparane sulphate HS I on biological or artificialsurfaces effects that suchlike coated surfaces get blood compatible andsuitable for the permanent blood contact. A disadvantage whereas is,that this process for the preparation of HS I premises the cultivationof endothelic cells, so that the economical suitability of this processis strongly limited, because the cultivation of endothelic cells is timetaking and greater amounts of cultivated endothelic cells are onlyobtainable with immense expenditure.

[0016] The present invention solves the object by providing medicaldevices that show properties of a surface coating of determinedpolysaccharides and paclitaxel. Instead of or together with paclitaxeldetermined other antiphlogistic as well as anti-inflammatory drugs resp.agent combinations of simvastatine,2-methylthiazolidine-2,4-dicarboxylic acid and the correspondent sodiumsalt), macrocyclic suboxide (MCS) and its derivatives, tyrphostines,D24851, thymosin a-1, interleucine-1β inhibitors, activated protein C(aPC), MSH, fumaric acid and fumaric acid ester, PETN (pentaerythritoltetranitrate), PI88, dermicidin, baccatin and its derivatives, docetaxeland further derivatives of paclitaxel, tacrolimus, pimecrolimus,trapidil, a- and β-estradiol, sirolimus, colchicin, andmelanocyte-stimulating hormone (α-MSH) can be used. Methods for theproduction of these hemocompatible surfaces are given in the claims20-31. Preferred embodiments can be found in the dependent claims, theexamples as well as the figures.

[0017] The subject matter of the present invention are medical devicesthe surface of which is at least partially covered with a hemocompatiblelayer, wherein the hemocompatible layer comprises at least one compoundof the formula 1:

[0018] wherein

[0019] n is an integer between 4 and 1050 and

[0020] Y represents the residues —CHO, —COCH₃, —COC₂H₅, —COC₃H₇,—COC₄H₉, —COC₅H₁₁, —COCH(CH₃)₂, —COCH₂CH(CH₃)₂, —COCH(CH₃)C₂H₅,—COC(CH₃)₃, —CH₂COO⁻, —C₂H₄COO⁻, —C₃H₆COO⁻, —C₄H₈COO⁻.

[0021] It is also possible to use any salts of the compounds offormula 1. The hemocompatible layer can be added directly onto thesurface of a preferably non hemocompatible medical device or depositedonto other biostable and/or biodegradable layers. Further on additionalbiostable and/or biodegradable and/or hemocompatible layers can belocalised on the hemocompatible layer. In addition to this the activeagent paclitaxel is present on, in and/or under the hemocompatible layeror the hemocompatible layers, respectively. The active agent(paclitaxel) can form herein an own active agent layer on or under thehemocompatible layer and/or can be incorporated in at least one of thebiostable, biodegradable and/or hemocompatible layers. Preferably thecompounds of the general formula 1 are used, wherein Y is one of thefollowing groups: —CHO, —COCH₃, —COC₂H₅ or —COC₃H₇. Further on preferredare the groups —CHO, —COCH₃, —COC₂H₅ and especially preferred is thegroup —COCH₃.

[0022] The compounds of the general formula 1 contain only a smallamount of free amino groups. Because of the fact that with theninhydrine reaction free amino groups could not be detected anymore, dueto the sensitivity of this test it can be implied that less than 2%,preferred less than 1% and especially preferred less than 0.5% of all—NH—Y groups are present as free amino groups, i.e. within this lowpercentage of the —NH—Y groups Y represents hydrogen.

[0023] Because polysaccharides of the general formula 1 containcarboxylate groups and amino groups, the general formula covers alkalias well as alkaline earth metal salts of the correspondingpolysaccharides. Alkali metal salts like the sodium salt, the potassiumsalt, the lithium salt or alkaline earth metal salts like the magnesiumsalt or the calcium salt can be mentioned. Further on with ammonia,primary, secondary, tertiary and quaternary amines, pyridine andpyridine derivatives ammonium salts, preferably alkylammonium salts andpyridinium salts can be formed. Among the bases, which form salts withthe polysaccharides, are inorganic and organic bases as for exampleNaOH, KOH, LiOH, CaCO₃, Fe(OH)₃, NH₄OH, tetraalkylammonium hydroxide andsimilar compounds.

[0024] The polysaccharides according to formula 1 possess molecularweights from 2 kD to 15 kD, preferred from 4 kD to 13 kD, more preferredfrom 6 kD to 12 kD and especially preferred from 8 kD to 11 kD. Thevariable n is an integer in the range of 4 to 1050. Preferred n is aninteger from 9 to 400, more preferred an integer from 14 to 260 andespecially preferred an integer between 19 and 210.

[0025] The general formula 1 shows a disaccharide, which has to beviewed as the basic module for the used polysaccharides and that formesthe polysaccharide by the n-fold (multiple) sequencing of the basicmodule. This basic module which is built of two sugar molecules shallnot be interpreted in the manner, that the general formula 1 onlyincludes polysaccharides with an even number of sugar molecules. Theformula implements of course also polysaccharides with an odd number ofsugar building units. The end groups of the polysaccharides arerepresented by hydroxyl groups.

[0026] Especially preferred are medical devices which containimmediately on the surface of the medical device a hemocompatible layerconsisting of the compounds according to formula 1 and above it a layerof paclitaxel. The paclitaxel layer can diffuse partially into thehemocompatible layer or get taken up totally by the hemocompatiblelayer.

[0027] It is further preferred, if at least one biostable layer ispresent under the hemocompatible layer. In addition the hemocompatiblelayer can be coated totally and/or partially with at least one more,above lying biostable and/or biodegradable layer. Preferred is anexternal biodegradable or hemocompatible layer.

[0028] A further preferred embodiment contains a layer of paclitaxelunder the hemocompatible layer or between the biostable and thehemocompatible layer, so that paclitaxel is released slowly through thehemocompatible layer. Paclitaxel can be bound covalently and/oradhesively in and/or on the hemocompatible layer and/or the biostableand/or the biodegradable layer, in which the adhesive bonding ispreferred.

[0029] As biodegradable substances for the biodegradable layer(s) can beused: polyvalerolactones, poly-ε-decalactones, polylactonic acid,polyglycolic acid, polylactides, polyglycolides, copolymers of thepolylactides and polyglycolides, poly-ε-caprolactone,polyhydroxybutanoic acid, polyhydroxybutyrates, polyhydroxyvalerates,polyhydroxybutyrate-co-valerates, poly(1,4-dioxane-2,3-diones),poly(1,3-dioxane-2-one), poly-para-dioxanones, polyanhydrides such aspolymaleic anhydrides, polyhydroxymethacrylates, fibrin,polycyanoacrylates, polycaprolactonedimethylacrylates, poly-b-maleicacid, polycaprolactonebutyl-acrylates, multiblock polymers such as e.g.from oligocaprolactonedioles and oligodioxanonedioles, polyether estermultiblock polymers such as e.g. PEG and poly(butyleneterephtalates),polypivotolactones, polyglycolic acid trimethyl-carbonates,polycaprolactone-glycolides, poly(g-ethylglutamate),poly(DTH-iminocarbonate), poly(DTE-co-DT-carbonate),poly(bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic acidtrimethyl-carbonates, polytrimethylcarbonates, polyiminocarbonates,poly(N-vinyl)-pyrrolidone, polyvinylalcoholes, polyesteramides,glycolated polyesters, polyphosphoesters, polyphosphazenes,poly[p-carboxyphenoxy)propane], polyhydroxypentanoic acid,polyanhydrides, polyethyleneoxide-propyleneoxide, soft polyurethanes,polyurethanes with amino acid residues in the backbone, polyether esterssuch as polyethyleneoxide, polyalkeneoxalates, polyorthoesters as wellas their copolymers, lipides, carrageenans, fibrinogen, starch,collagen, protein based polymers, polyamino acids, synthetic polyaminoacids, zein, modified zein, polyhydroxyalkanoates, pectic acid, actinicacid, modified and non modified fibrin and casein,carboxymethylsulphate, albumin, moreover hyaluronic acid, chitosan andits derivatives, heparansulphates and its derivatives, heparins,chondroitinsulphate, dextran, b-cyclodextrins, copolymers with PEG andpolypropyleneglycol, gummi arabicum, guar, gelatine, collagen,collagen-N-hydroxysuccinimide, lipids, phospholipids, modifications andcopolymers and/or mixtures of the afore mentioned substances.

[0030] As biostable substances for the biostable layer(s) can be used:polyacrylic acid and polyacrylates as polymethylmethacrylate,polybutylmethacrylate, polyacrylamide, polyacrylonitriles, polyamides,polyetheramides, polyethylenamine, polyimides, polycarbonates,polycarbourethanes, polyvinylketones, polyvinylhalogenides,polyvinylidenhalogenides, polyvinylethers, polyisobutylenes,polyvinylaromates, polyvinylesters, polyvinylpyrollidones,polyoxymethylenes, polytetramethyleneoxide, polyethylene, polypropylene,polytetrafluoroethylene, polyurethanes, polyetherurethanes,silicone-polyetherurethanes, silicone-polyurethanes,silicone-polycarbonate-urethanes, polyolefine elastomeres,polyisobutylenes, EPDM gums, fluorosilicones, carboxymethylchitosanes,polyaryletheretherketones, polyetheretherketones,polyethylenterephthalate, polyvalerates, carboxymethylcellulose,cellulose, rayon, rayontriacetates, cellulosenitrates,celluloseacetates, hydroxyethylcellulose, cellulosebutyrates,celluloseacetatebutyrates, ethylvinylacetate copolymers, polysulphones,epoxy resins, ABS resins, EPDM gums, silicones as polysiloxanes,polydimethylsiloxanes, polyvinylhalogenes and copolymers,celluloseethers, cellulosetriacetates, chitosanes and copolymers and/ormixtures of these substances.

[0031] It is possible to furnish any medical devices with the hereindisclosed hemocompatible surfaces, especially those, which shall besuitable for the short- or the longterm contact with blood or bloodproducts. Such medical devices are for example prostheses, organs,vessels, aortas, heart valves, tubes, organ spareparts, implants,fibers, hollow fibers, stents, hollow needles, syringes, membranes,tinned goods, blood containers, titrimetric plates, pacemakers,adsorbing media, chromatography media, chromatography columns,dialyzers, connexion parts, sensors, valves, centrifugal chambers,recuperators, endoscopes, filters, pump chambers. The present inventionis especially related to stents.

[0032] The polysaccharides of formula 1 can be formed from heparinand/or heparansulphates. These materials are in structurally view quitesimilar compounds. Heparansulphates occur ubiquitously on cell surfacesof mammals. In dependence from the cell type they differ strongly inmolecular weight, degree of acetylation and degree of sulphation.Heparansulphate from liver shows for example an acetylation coefficientof about 50%, whereas the heparansulphate of the glycocalix fromendothelic cells can exhibit an acetylation coefficient from about 90%and higher. Heparin shows only a quite low degree of acetylation fromabout up to 5%. The sulphation coefficient of the heparansulphate fromliver and of heparin is ˜2 per disaccharide unit, in case ofheparansulphate from endothelial cells close to 0 and inheparansulphates from other cell types between 0 and 2 per disaccharideunit.

[0033] The compounds of the general formula 1 are characterized by anamount of sulphate groups per disaccharide unit of less than 0.05.Further on the amount of free amino groups in these compounds is lessthan 1% based on all —NH—Y groups.

[0034] The following image shows a tetrasaccharide unit of a heparin ora heparansulphate with random orientation of the sulphate groups andwith a sulphation coefficient of 2 per disaccharide unit as it istypical for heparin:

[0035] All heparansulphates have with heparin a common sequence inbiosynthesis. First of all the core protein with the xylose-containingbonding region is formed. It consists of the xylose and two galactoseresidues connected to it. To the last of the two galactose units aglucuronic acid and a galactosamine is connected alternately until theadequate chain length is reached. Finally, a several step enzymaticmodification of this common polysaccharide precursor of allheparansulphates and of heparin follows by means of sulphotransferasesand epimerases which generate by their varying completeness oftransformation the broad spectra of different heparansulphates up toheparin.

[0036] Heparin is alternately build of D-glucosamine and D-glucuronicacid resp. L-iduronic acid, in which the amount of L-iduronic acid is upto 75%. D-glucosamine and D-glucuronic acid are connected in aβ-1,4-glycosidic resp. L-iduronic acid in an a-1,4-glycosidic bonding tothe disaccharide, that forms the heparin subunits. These subunits areagain connected to each other in a β-1,4-glycosidic way and lead toheparin. The position of the sulphonyl groups is variable. In averageone tetrasaccharide unit contains 4 to 5 sulphuric acid groups.Heparansulphate, also named as heparitinsulphate, contains withexception of the heparansulphate from liver less N- and O-boundsulphonyl goups as heparin but in exchange more N-acetyl goups. Theamount of L-iduronic acid compared to heparin is also lower.

[0037] As it is evident from FIG. 1 the compounds of the general formula(cf. FIG. 1b as example) are structurally similar to the naturalheparansulphate of endothelial cells, but avoid the initially mentioneddisadvantages by the use of endothelial cell heparan sulphates.

[0038] For the antithrombotic activity a special pentasaccharide unit ismade responsible, which can be found in commercial heparin preparativesin about every 3^(rd) molecule. Heparin preparations of differentantithrombotic activity can be produced by special separationtechniques. In highly active, for example byantithrombin-III-affinitychromatography obtained preparations(“High-affinity”-heparin) this active sequence is found in every heparinmolecule, while in “No-affinity”-preparations no characteristicalpentasaccharide sequences and thus no active inhibition of coagulationcan be detected. Via interaction with this pentasaccharide the activityof antithrombin III, an inhibitor of the coagulation key factorthrombin, is essentially exponentiated (bonding affinity increase up tothe factor 2×10³) [Stiekema J. C. J.; Clin Nephrology 26, Suppl. Nr 1,S3-S8, (1986)].

[0039] The amino groups of the heparin are mostly N-sulphated orN-acetylated. The most important O-sulphation positions are the C2 inthe iduronic acid as well as the C6 and the C3 in the glucosamine. Forthe activity of the pentasaccharide onto the plasmatic coagulationbasically the sulphate group on C6 is made responsible, in smallerproportion also the other functional groups.

[0040] Surfaces of medicinal implants coated with heparin orheparansulphates are and remain only conditionally hemocompatible by thecoating. The heparin or heparansulphate which is added onto theartificial surface loses partially in a drastic measure itsantithrombotic activity which is related to a restricted interaction dueto steric hindrence of the mentioned pentasaccharide units withantithrombin III. Because of the immobilisation of these polyanionicsubstances a strong adsorption of plasma protein on the heparinatedsurface is observed in all cases what eliminates on the one hand thecoagulation suppressing effect of heparin resp. of heparansulphates andinitialises on the other hand specific coagulation processes by adherentand hereby tertiary structure changing plasma proteins (e.g. albumin,fibrinogen, thrombin) and hereon adherent platelets.

[0041] Thus a correlation exists on the one hand between the limitedinteraction of the pentasaccharide units with antithrombin III byimmobilisation on the other hand depositions of plasma proteins on theheparin-resp. heparansulphate layer on the medicinal implant take place,which leads to the loss(es) of the antithrombotic properties of thecoating and which can even turn into the opposite, because the plasmaprotein adsorption, that occurs during a couple of seconds leads to theloss of the anticoagulational surface and the adhesive plasma proteinschange their tertiary structure, whereby the antithrombogenity of thesurface turns vice versa and a thrombogenous surface arises.Surprisingly it could be detected, that the compounds of the generalformula 1, despite of the structural differences to the heparin resp.heparansulphate, still show the hemocompatible properties of heparin andadditionally after the immobilisation of the compounds no noteworthydepositions of plasma proteins, which represent an initial step in theactivation of the coagulation cascade, could be observed. Thehemocopatible properties of the compounds according to invention stillremain also after their immobilisation on artificial surfaces.

[0042] Further on it is supposed that the sulphate groups of the heparinresp. the heparansulphates are necessary for the interaction withantithrombin III and impart thereby the heparin resp. theheparansulphate the anticoagulatory effect. The inventive compounds arenot actively coagulation suppressive, i.e. anticoagulative, due to analmost complete desulphation the sulphate groups of the compounds areremoved up to a low amount of below 0.2 sulphate groups per disaccharideunit.

[0043] The inventive compounds of the general formula 1 can be generatedfrom heparin or heparansulphates by first substantially completedesulphation of the polysaccharide and subsequently substantiallycomplete N-acylation. The term “substantially completely desulphated”refers to a desulphation degree of above 90%, preferred above 95% undespecially preferred above 98%. The desulphation coefficient can bedetermined according to the so-called ninhydrin test which indicatesfree amino groups. The desulphation takes place in the way as with DMMB(dimethylmethylene blue) no colour reaction is obtained. This colourtest is suitable for the indication of sulphated polysaccharides but itsdetection limit is not known in technical literature. The desulphationcan be carried out for example by pyrolysis of the pyridinium salt in asolvent mixture. Especially a mixture of DMSO, 1,4-dioxane and methanolhas proven of value.

[0044] Heparansulphates as well as heparin were desulphated via totalhydrolysis and subsequently reacylated. Thereafter the number ofsulphate groups per disaccharide unit (S/D) was determined by ¹³C-NMR.The following table 1 shows these results on the example of heparin anddesulphated, reacetylated heparin (Ac-heparin). TABLE 1 Distribution offunctional groups per disaccharide unit on the example of heparin andAc-heparin as determined by ¹³C-NMR-measurements. 2-S 6-S 3-S NS N—AcNH₂ S/D Heparin 0.63 0.88 0.05 0.90 0.08 0.02 2.47 Ac-heparin 0.03 0 0 01.00 — 0.03

[0045] A sulphate content of about 0.03 sulphate groups/disaccharideunit (S/D) in case of Ac-heparin in comparison with about 2.5 sulphategroups/disaccharide unit in case of heparin was reproducibly obtained.

[0046] As described above the difference in the sulphate contents ofheparin resp. heparansulphates has a considerable influence on theactivity adverse to antithrombin III and the coagulatory effects ofthese compounds. These compounds have a content of sulphate groups perdisaccharide unit of less than 0.2, preferred less than 0.07, morepreferred less than 0.05 and especially preferred less than 0.03sulphate groups per disaccharide unit.

[0047] By the removal of the sulphate groups of heparin, to which theactive coagulation suppressive working mechanism is accredited to, onereceives for a surface refinement suitable hemocompatible, coagulationinert oligo-resp. polysaccharide which on the one hand has no activerole in the coagulation process and which on the other hand is notdetected by the coagulation system as foreign surface. Accordingly thiscoating imitates successfully the nature given highest standard ofhemocompatibility and passivity against the coagulation activecomponents of the blood. The examples 3 and 4 clarify, that surfaces,which are coated with the compounds according to invention, especiallyare coated covalently, result in a passivative, athrombogeneous andhemocompatible coating. This is definitely proven by the example ofAc-heparins.

[0048] Substantially completely N-acylated refers to a degree ofN-acylation of above 94%, preferred above 97% and especially preferredabove 98%. The acylation runs in such a way completely that with theninhydrin reaction for detection of free amino groups no colour reactionis obtained anymore. As acylation agents are preferably used carboxylicacid chlorides, -bromides or -anhydrides. Acetic anhydride, propionicanhydride, butyric anhydride, acetic acid chloride, propionic acidchloride or butyric acid chloride are for example suitable for thesynthesis of the compounds according to invention. Especially suitableare carboxylic anhydrides as acylation agents.

[0049] As solvent especially for carboxylic acid anhydrides deionisedwater is used, especially together with a cosolvent which is added in anamount from 10 to 30 volume percent. As cosolvents are suitablemethanol, ethanol, DMSO, DMF, acetone, dioxane, THF, ethyl acetate andother polar solvents. In case of the use of carboxylic acid halogenidespreferably polar water free solvents such as DMSO or DMF are used.

[0050] The inventive compounds of the general formula comprise in thehalf of the sugar molecules a carboxylate group and in the other half aN-acyl group.

[0051] The present invention describes the use of the compounds with thegeneral formula 1 as well as salts of these compounds for the coating,especially a hemocompatible coating of natural and/or artificialsurfaces. Under “hemocompatible” the characteristic of the compoundsaccording to invention is meant, not to interact with the compounds ofthe blood coagulation system or the platelets and so not to initiate theblood coagulation cascade.

[0052] In addition the invention reveals polysaccharides for thehemocompatible coating of surfaces. Preferred are polysaccharides in therange of the above mentioned molecular weight limits. The usedpolysaccharides are characterised in that they contain the sugarbuilding unit N-acylglucosamine in a great amount. This means that 40 to60% of the sugar building units are N-acylglucosamine and substantiallythe remaining sugar building units bear each a carboxyl group. Thepolysaccharides consist generally in more than 95%, preferred in morethan 98%, of only two sugar building units, whereas one sugar buildingunit bears a carboxyl group and the other one a N-acyl group.

[0053] One sugar building unit of the polysaccharides isN-acylglucosamine preferred N-acetylglucosamine and in case of the otherone it is the uronic acids glucuronic acid and iduronic acid. Preferredare polysaccharides, which conspire substantially the sugar glucosamine,whereas substantially the half of the sugar building units bears aN-acyl group, preferred a N-acetyl group, and the other half of theglucosamine building units bears one carboxyl group which is bonddirectly by the amino group or by one or more methylenyl groups. In thecase of these carboxylic acid groups bound to the amino group it isconcerned to be preferred the carboxymethyl- or carboxyethyl groups.Furthermore, polysaccharides are preferred which substantially conspirein one half of N-acylglucosamine, preferred of N-acetylglucosamine andsubstantially conspire in the other half of the uronic acids glucuronicacid and iduronic acid. Especially preferred are the polysaccharides,that show a substantially alternating sequence of N-acylglucosamine andone of the both uronic acids.

[0054] Surprisingly it was shown, that for the applications according toinvention especially desulphated and substantially N-acylated heparin isespecially suitable. Especially N-acetylated heparin is suitable for thehemocompatible coating.

[0055] The term “substantially” shall make clear, that statisticalvariations are to be taken into account. One substantially alternatingsequence of the sugar building units implies, that generally no twoequal sugar building units are bound to each other but does not excludetotally such a defect connection. In accordance “substantially the half”means almost 50% but allows small variations, because especially in thecase of biosynthetically synthesised macromolecules the ideal case isnever reached and some variations are always to be taken into account,because enzymes do not work perfectly and in catalysis always some errorrate has to be anticipated. Whereas in case of natural heparin astrongly alternating sequence of N-acetylglucosamine and the uronic acidunits is existing.

[0056] Furthermore, methods for hemocompatible coating of surfaces aredisclosed which are especially destined for the direct blood contact. Incase of these methods a natural and/or artificial surface is providedand the above described polysaccharides are immobilised on this surface.

[0057] The immobilisation of the polysaccharides on these surfaces canbe achieved via hydrophobic interactions, van der Waals forces,electrostatic interactions, hydrogen bonds, ionic interactions,cross-linking of the polysaccharides and/or by covalent bonding onto thesurface. Preferred is the covalent linkage of the polysaccharides(side-on bonding), more preferred the covalent single-point linkage(side-on bonding) and especially preferred the covalent end-pointlinkage (end-on bonding).

[0058] In the following the coating methods according to invention aredescribed.

[0059] Biological and/or artificial surfaces of medical devices can beprovided with a hemocompatible coating by means of the following method:

[0060] a) providing a surface of a medical device and

[0061] b) deposition of at least one compound of the general formula 1according to claim 1 as hemocompatible layer onto this surface and/or

[0062] b′) deposition of a biostable and/or biodegradable layer onto thesurface of the medical device or the hemocompatible layer.

[0063] “Deposition” shall refer to at least partial coating of a surfacewith the adequate compounds, wherein the compounds are positioned and/orimmobilised or anyhow anchored on and/or in the subjacent surface.

[0064] Under “substantially the remaining sugar building units” is to beunderstood that 93% of the remaining sugar building units, preferred 96%and especially preferred 98% of the remaining 60%-40% of the sugarbuilding units bear a carboxyl group.

[0065] An uncoated and/or non hemocompatible surface is preferablyprovided. “Non hemocompatible” surfaces shall refer to such surfacesthat can activate the blood coagulatory system, thus are more or lessthrombogeneous.

[0066] An alternative embodiment comprises the steps:

[0067] a) providing surface of a medical device and

[0068] b) deposition of at least one inventive polysaccharide accordingto formula 1,

[0069] b′) deposition of a biostable layer onto the surface of themedical device and

[0070] d′) deposition of a further hemocompatible layer of at least oneinventive polysaccharide according to formula 1.

[0071] The last-mentioned embodiment makes sure, even in the case ofe.g. mechanical damage of the polymeric layer and therewith also of theexterior hemocompatible layer, that the surface coating does not loseits characteristic of being blood compatible.

[0072] Under “biological or artificial” surface is the combination of anartificial medical device with an artificial part to be understood, e.g.pork heart with an artificial heart valve.

[0073] The single layers are deposited preferably by dipping or sprayingmethods, whereas one can deposit also paclitaxel at the same time withthe deposition of one layer onto the medical device surface, which isthen implemented in the respective layer covalently and/or adhesivelybound. In this way it is possible at the same time with the depositionof a hemocompatible layer onto the medical device to deposit the activeagent paclitaxel. The substances for the biostable or biodegradablelayers were itemised already above.

[0074] Onto this first biostable and/or biodegradable or hemocompatiblelayer it is then possible in an additional non compulsory step c) todeposit an agent layer of paclitaxel. In a preferred embodimentpaclitaxel is bound covalently on the subjacent layer. Also paclitaxelis preferably deposited by dipping or spraying methods on and/or in thehemocompatible layer or the biostable layer.

[0075] After the step b) or the step c) an additional step d) can followwhich implements the deposition of at least one biodegradable layerand/or at least one biostable layer onto the hemocompatible layer resp.the layer of paclitaxel.

[0076] According to the alternative embodiments after step b′) or stepc) a step d′) can follow which implements the deposition of at least onecompound of the general formula 1 as hemocompatible layer onto thebiostable and/or biodegradable layer resp. the layer of paclitaxel.Preferably after step b′) the step d′) follows.

[0077] After step d) resp. d′) the deposition of paclitaxel can takeplace into and/or onto the at least one biodegradable and/or biostablelayer or the hemocompatible layer.

[0078] The single layers as well as paclitaxel are preferably depositedand/or implemented by dipping or spraying methods onto and/or into thesubjacent layer.

[0079] According to a preferred embodiment the biostable layer isdeposited on the surface of the medical device and completely orincompletely covered with a hemocompatible layer which (preferablycovalently) is bound to the biostable layer.

[0080] Preferably the hemocompatible layer comprises heparin of nativeorigin of regioselectively synthesised derivatives of differentsulphation coefficients (sulphation degrees) and acylation coefficients(acylation degrees) in the molecular weight range of thepentasaccharide, which is responsible for the antithrombotic activity,up to the standard molecular weight of the purchasable heparin of 13 kD,heparansulphate and its derivatives, oligo- and polysaccharides of theerythrocytic glycocalix, desulphated and N-reacetylated heparin,N-carboxymethylated and/or partially N-acetylated chitosan as well asmixtures of these substances.

[0081] Subject of the invention are also medical devices which arehemocompatibly coated according to one of the herein mentioned methods.In the case of the medical devices it is preferably a matter of stents.

[0082] The conventional stents, which can be coated according to theinventive methods, consist of stainless steel, nitinol or other metalsand alloys or of synthetic polymers.

[0083] The stents according to invention are coated with an according tothe general formula 1 preferred covalently bound hemocompatible layer. Asecond layer covers this first hemocompatible layer completely or alsoincompletely. This second layer conspires preferably paclitaxel. Thehemocompatible coating of a stent provides on the one hand the necessaryblood compatibility and reduces so the risk of thrombosis and also thecontainment of inflammation reactions due to the intrusion and theabsence of a non-endogenous surface, and paclitaxel, which is preferredto be distributed homogeneously over the total surface of the stentprovides that the covering of the stent surface with cells, especiallysmooth muscle and endothelial cells, takes place in a controlled way, sothat the interplay of thrombosis reactions and inflammation reactions,the release of growth factors, proliferation and migration of cellsduring the recovery process provides the generation of a novel“repaired” cell layer, which is referred to as neointima.

[0084] Thus, the use of paclitaxel, covalently or/and adhesively boundto the subjacent layer or/and covalently or/and adhesively implementedin at least one layer, ensures, that this active agent is set freecontinuously and in small doses, so that the population of the stentsurface by cells is not inhibited, however an excessive population andthe ingrowth of cells into the vessel lumen is prevented. Thiscombination of both effects awards the ability to the stent according toinvention, to grow rapidly into the vessel wall and reduces both therisk of restenosis and the risk of thrombosis. The release of paclitaxelspans about a period from 1 to 12 months, preferably 1 to 3 months afterimplantation.

[0085] Paclitaxel is preferred contained in a pharmaceutical activeconcentration from 0.001-10 mg per cm² stent surface, preferred 0.01-5mg and especially preferred 0.1-1.0 mg per cm² stent surface. Additionalactive agents can be contained in similar concentration in the same orin the hemocompatible layer.

[0086] The applied amounts of polymer are per layer between 0.01 mg to 3mg, preferred 0.20 mg to 1 mg and especially preferred between 0.2 mg to0.5 mg. Suchlike coated stents release the active agent paclitaxelcontrolled and continuously and hence are excellently suitable for theprevention and reduction of restenosis.

[0087] These stents with a hemocompatible coating are generated, as oneprovides stents and deposits preferred covalently one hemocompatiblelayer according to the general formula, which masks the surface of theimplantate permanently after the release of the active agent and soafter the decay of the active agent influence.

[0088] The preferred embodiment of the stents according to inventionshows a coating, which consists of at least two layers. Thereby named assecond layer is that layer, which is deposited on the first layer.According to the two-layer design the first layer conspires thehemocompatible layer, which is substantially completely covered by asecond layer, which consists of paclitaxel, that is covalently and/oradhesively bound to the first layer.

[0089] The paclitaxel layer is dissolved slowly, so that the activeagent is released according to the velocity of the solution process. Thefirst hemocompatible layer guarantees the necessary blood compatibilityof the stent in the degree as the active agent is removed. By therelease of the active agent the adhesion of cells is strongly reducedonly for a certain period of time and an aimed controlled adhesion isenabled, where the external layer had been already widely degradated.Finally the hemocompatible layer remains as athrombogeneous surface andmasks the foreign surface in such a way, that no life-threateningreaction can occur anymore.

[0090] Suchlike stents can be generated by a method of thehemocompatible coating of stents, to which the following principleunderlies:

[0091] a. providing of a stent

[0092] b. deposition of a preferred covalently bound hemocompatiblelayer

[0093] c. Substantially complete covering of the hemocompatible layer bya dipping or spraying method with the antiproliferative active agentpaclitaxel.

[0094] The stents according to invention solve both the problem of acutethrombosis and the problem of neointima hyperplasia after a stentimplantation. In addition the inventive stents are especially wellsuited, because of their coating for the continuous release of one ormore antiproliferative, immuno-suppressive active agents. Due to thiscapability of the aimed continuous active agent release in a requiredamount the inventively coated stents prevent the danger of restenosisalmost completely.

[0095] The natural and/or artificial surfaces which had been coatedaccording to the above described method with a hemocompatible layer ofaforesaid polysaccharides, are suitable especially as implants resp.organ replacement parts, that are in direct contact with the bloodcircuit and blood, preferably in the form of stents in combination withan antiproliferative active agent, preferably paclitaxel, for theprevention of restenosis.

[0096] The inventively coated medical devices are suited especially butnot only for the direct and permanent blood contact, but showsurprisingly also the characteristic to reduce or even to prevent theadhesion of proteins onto suchlike coated surfaces. The adhesion ofplasma proteins on foreign surfaces which come in contact with blood isan essential and initial step for the further events concerning therecognition and the implementing action of the blood system.

[0097] This is for example important in the in vitro diagnostics frombody fluids. Thus the deposition of the inventive coating prevents or atleast reduces for example the unspecific adhesion of proteins onmicro-titer plates or other support mediums which are used fordiagnostic detection methods, that disturb the generally sensitive testreactions and can lead to a falsification of the analysis result.

[0098] By use of the coating according to invention on adsorption mediaor chromatography media the unspecific adhesion of proteins is alsoprevented or reduced, whereby better separations can be achieved andproducts of greater purity can be generated.

DESCRIPTION OF FIGURES

[0099]FIG. 1 shows a tetrasaccharide unit of a heparin orheparansulphate with statistic distribution of the sulphate groups and asulphation coefficient of 2 per disaccharide unit as it is typical forheparin (FIG. 1a). For comparison of the structural similarities FIG. 1bshows an example of a compound according to the general formula in thedescription.

[0100]FIG. 2 shows the influence of an into a PVC-tube expanded, surfacemodified stainless steel coronary stent on the platelet loss (PLT-loss).

[0101] An uncoated stainless steel coronary stent was measured asreference. As zero value the level of the platelet loss in case of thePVC-tube without stainless steel coronary stent was set.

[0102] Thereby SH1 is a with heparin covalently coated stent, SH2 is awith chondroitinsulphate coated stent; SH3 is a stent coated withpolysaccharides gained from the erythrocytic glycocalix and SH4 is awith Ac-heparin covalently coated stainless steel coronary stent.

[0103]FIG. 3 shows a schematic presentation of the restenosis rate ofwith completely desulphated and N-reacetylated heparin (Ac-heparin)covalently coated stents and with oligo- and polysaccharides of theerythrocytic glycocalix (polysacch. of erythro. glycoc.) coated stentsin comparison to the uncoated stent and with polyacrylic acid (PAS)coated stents after 4 weeks of implantation time in pork.

[0104]FIG. 4 quantitative coronary angiography:

[0105] Images of the cross sections through the stent containingvessel-segment of one with Ac-heparin coated stent (a.) and ascomparison of one uncoated (unco. or bare) stent (b.). After four weeksin the animal experiment (pork) a clear difference in the thicknesses ofthe formed neointimas can be observed.

[0106]FIG. 5 elution plot of paclitaxel from the stent (without supportmedium).

EXAMPLES Example 1

[0107] Synthesis of Desulphated Reacetylated Heparin:

[0108] 100 ml amberlite IR-122 cation exchange resin were added into acolumn of 2 cm diameter, with 400 ml 3M HCl in the H⁺-form converted andrinsed with distilled water, until the eluate was free of chloride andpH neutral. 1 g sodium-heparin was dissolved in 10 ml water, added ontothe cation exchange column and eluted with 400 ml of water. The eluatewas added dropvise into a receiver with 0.7 g pyridine and afterwardstitrated with pyridine to pH 6 and freeze-dried.

[0109] 0.9 g heparin-pyridinium-salt were added in a round flask with areflux condenser with 90 ml of a 6/3/1 mixture ofDMSO/1,4-dioxan/methanol (v/v/v) and heated for 24 hours to 90° C. Then823 mg pyridinium chloride were added and heated additional 70 hours to90° C. Afterwards it was diluted with 100 ml of water and titrated withdilute sodium hyrdoxide to pH 9. The desulphated heparin was dialyzedcontra water and freeze-dried.

[0110] 100 mg of the desulphated heparin were solved in 10 ml of water,cooled to 0° C. and added with 1.5 ml methanol under stirring. To thissolution were added 4 ml dowex 1×4 anion exchange resin in the OH⁻-formand afterwards 150 μl of acetic anhydride and stirred for 2 hours at 4°C. Then the resin was removed by filtration and the solution wasdialyzed contra water and freeze-dried.

Example 2

[0111] Synthesis of Desulphated N-Propionylated Heparin:

[0112] 100 ml amberlite IR-122 cation exchange resin were added into acolumn of 2 cm diameter, with 400 ml 3M HCl in the H⁺-form converted andrinsed with distilled water, until the eluate was free of chloride andpH neutral. 1 g sodium-heparin was dissolved in 10 ml water, added ontothe cation exchange column and eluted with 400 ml of water. The eluatewas added dropvise into a receiver with 0.7 g pyridine and afterwardstitrated with pyridine to pH 6 and freeze-dried.

[0113] 0.9 g heparin-pyridinium-salt were added in a round flask with areflux condenser with 90 ml of a 6/3/1 mixture ofDMSO/1,4-dioxan/methanol (v/v/v) and heated for 24 hours to 90° C. Then823 mg pyridiniumchloride were added and heated additional 70 hours to90° C. Afterwards it was diluted with 100 ml of water and titrated withdilute sodium hydroxide to pH 9. The desulphated heparin was dialyzedcontra water and freeze-dried.

[0114] 100 mg of the desulphated heparin were solved in 10 ml of water,cooled to 0° C. and added with 1.5 ml methanol under stirring. To thissolution were added 4 ml dowex 1×4 anion exchange resin in the OH⁻-formand afterwards 192 μl of propionic anhydride and stirred for 2 hours at4° C. Then the resin was removed by filtration and the solution wasdialyzed contra water and freeze-dried.

Example 3

[0115] Hemocompatibility Measurements of Compounds According to theGeneral Formula 1 by ISO 10933-4 (In Vitro Measurements):

[0116] For the measurement of the hemocompatibility of the compoundsaccording to formula 1 cellulose membranes, silicon tubes and stainlesssteel stents were covalently coated with a compound according to formula1 and tested contra heparin as well as contra the corresponding, in thesingle tests utilised uncoated material surfaces.

[0117] 3.1. Cellulose Membranes (Cuprophan) Coated with Desulphated,Reacetylated Heparin (Ac-Heparin)

[0118] For the examination of the coagulatory physiologic interactionsbetween citrated whole blood and the Ac-heparin-resp. heparin-coatedcuprophan membranes the open perfusion system of theSakariassen-modified Baumgartner-chamber is used [Sakariassen K. S. etal.; J. Lab. Clin. Med. 102: 522-535 (1983)]. The chamber is composed offour building parts plus conical nipples and threaded joints and ismanufactured of polymethylmethacrylate and allows the parallelinvestigation of two modified membranes, so that in every run astatistic coverage is included. The construction of this chamber permitsquasi-laminar perfusion conditions.

[0119] After 5 minutes of perfusion at 37° C. the membranes areextracted and after fixation of the adherent platelets the plateletoccupancy is measured. The respective results are set into relation tothe highly thrombogeneous subendothelial matrix as negative standardwith a 100% platelet occupancy. The adhesion of the platelets takesplace secondary before the formation of the plasma protein layer on theforeign material. The plasma protein fibrinogen acts as cofactor of theplatelet aggregation. The such induced activation of the plateletsresults in the bonding of several coagulation associated plasmaproteins, as e.g. vitronectin, fibronectin and von Willebrand-factor onthe platelet surface. By their influence finally the irreversibleaggregation of the platelets occurs.

[0120] The platelet occupancy presents because of the describedinteractions an accepted quantum for the thrombogenity of surfaces incase of the foreign surface contact of blood. From this fact theconsequence arises: the lower the platelet occupancy is on the perfundedsurface the higher is the hemocompatibility of the examined surface tobe judged.

[0121] The results of the examined heparin-coated and Ac-heparin-coatedmembranes show clearly the improvement of the hemocompatibility of theforeign surface through the coating with Ac-heparin. Heparin-coatedmembranes show a 45-65% platelet occupancy, whilst Ac-heparin-coatedsurfaces show values from 0-5% (reference to subendothelial matrix with100% platelet occupancy).

[0122] The adhesion of the platelets on the Ac-heparinated surface isextremely aggravated due to the absent, for the activation of plateletsessential plasma proteins. Unlike to this the heparin-coated surfacewith the immediately incipient plasma protein adsorption offers optimalpreconditions for activation, deposition and aggregation of platelets,and ultimately the blood reacts with the corresponding defensemechanisms to the inserted foreign surface. Ac-heparin fulfills by farsuperior than heparin the requirements to the hemocompatibility of theforeign surface.

[0123] The interaction of plasma protein adsorption and plateletoccupancy as direct quantum for the thrombogenity of a surface, independence of the to the blood offered coating, is made clear especiallywell by this in-vitro test. Thus the utilisation of covalently boundheparin as antithrombotic operant surface is only strongly limited ornot possible at all. The interactions of immobilised heparin with bloodrevert themselves here into the undesired opposite—the heparin-coatedsurface gets thrombogeneous.

[0124] Obviously the outstanding importance of heparin as anantithrombotic is not transferable to covalently immobilised heparin. Inthe systemic application in dissolved form it can fully unfold itsproperties. But if heparin is not covalently immobilised, itsantithrombotic properties, if at all, is only short-lived. Different isthe Ac-heparin (“No-affinity”-heparin), that due to the desulphation andN-reacetylation in fact totally loses the active antithromboticproperties of the initial molecule, but acquires in return distinctiveathrombogeneous properties, that are demonstrably founded in thepassivity versus antithrombin III and the missing affinity towardscoagulation initiating processes and remain after covalent bonding.

[0125] Thereby Ac-heparin and thus the compounds of the general formula1 in total are optimally suitable for the camouflage of foreign surfacesin contact with the coagulation system.

[0126] 3.2. Immobilisation on Silicone

[0127] Through a 1 m long silicon tube with 3 mm inside diameter 100 mlof a mixture of ethanol/water 1/1 (v/v) was pumped in a circular motionfor 30 minutes at 40° C. Then 2 ml 3-(triethoxysilyl)-propylamine wereadded and pumped in a circular motion for additional 15 hours at 40° C.Afterwards it was rinsed in each case for 2 hours with 100 mlethanol/water and 100 ml water.

[0128] 3 mg of the deacetylated and reacetylated heparin (Ac-heparin)were dissolved at 4° C. in 30 ml 0.1 M MES-buffer pH 4.75 and mixed with30 mg CME-CDI (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulphonate). This solution was pumped in acircular motion for 15 hours at 4° C. through the tube. Afterwards itwas rinsed with water, 4 M NaCl solution and water in each case for 2hours.

[0129] 3.3 Determination of the Platelet Number (EN30993-4)

[0130] In a 1 m long silicone tube with 3 mm inside diameter two 2 cmlong formfitting glass tubes were placed. Then the tube was closed witha shrinkable tubing to a circle and filled under exclusion of air viasyringes with a 0.154 M NaCl solution. In doing so one syringe was usedto fill in the solution and the other syringe was used to remove theair. The solution was exchanged under exclusion of air (bleb-free) withthe two syringes against citrated whole blood of a healthy test person.Then the recess holes of the syringes were closed by pushing the glasstubes over them and the tube was clamped taut into a dialysis pump. Theblood was pumped for 10 minutes with a flow rate of 150 ml/min. Theplatelet content of the blood was measured before and after theperfusion with a coulter counter. For uncoated silicone tubes theplatelet loss was of 10%. In contrast to it the loss was in silicontubes, which were coated according to example 5.2, in average at 0%(number of experiments: n=3).

[0131] Also in this dynamic test system it is shown, that the activationof platelets on an Ac-heparin coated surface is reduced. Simultaneouslyit can be recorded, that the immobilisation of heparin executes anegative effect on the hemocompatibility of the utilised surface.Against it Ac-heparin shows, in accordance to its passive nature, noeffects in contact with the platelets.

[0132] 3.4 Whole Blood Experiments on 316 LVM Stainless Steel CoronaryStents

[0133] In line with the biocompatibility experiments 31 mm long 316 LVMstainless steel stents were covalently coated with Ac-heparin. In caseof a total surface of 2 cm² and a occupancy coefficient of about 20pm/cm² stent surface the charging of such a stent is about 0.35 μgAc-heparin. As comparison: in case of thrombosis prophylaxis the usualdaily application rate of heparin is in contrast 20-30 mg and thus wouldcorrespond to the at least 60.000 times the value.

[0134] These experiments were carried out with the establishedhemodynamic Chandler loop-system [A. Henseler, B. Oedekoven, C.Andersson, K. Mottaghy; KARDIOTECHNIK 3 (1999)]. Coated and uncoatedstents were expanded and tested in PVC tubes (medical grade PVC) with600 mm length and 4 mm inside diameter. The results of these experimentsconfirm the according to the silicone tubes discussed experiments. Theinitially to the stent attributed platelet loss in the perfusate of 50%is reduced by the refinement of the stent surface with Ac-heparin bymore than 80%.

[0135] The influence of in the tube expanded, surface modified coronarystents to the platelet loss is evaluated in further Chandler testsduring a 45 minute whole blood perfusion. For this primarily thestent-free PVC tube is analysed, the outcome of this is the zero value.The empty tube shows an average platelet loss of 27.4% regarding to thedonor blood at a standard aberration of solely 3.6%. This base valueunderlied different surface modified stents are expanded in the PVCtubes and are analysed under analogous conditions on the by them causedplatelet loss. It occurs also in this case, that the stent coveredsurface, which solely accounts for about 0.84% of the total testsurface, causes a significant and reproducable effect on the plateletcontent. According to the empty tube (base value) the analysis of thepolished, chemically not surface coated stent yields an additionalaverage platelet loss of 22.7%. Therewith causes this compared to thePVC empty tube less than 1% measurable foreign surface an approximatelycomparable platelet loss. A direct result is that the medicinalstainless steel 316 LVM used as stent material induces an about 100times stronger platelet damage compared to a medical grade PVC surface,although this test surface only accounts for 0.84% of the total surface.

[0136] The analysed surface coatings on the stainless steel coronarystents show to be able to reduce very clearly the enormous dimension ofthe stent induced platelet damage (see FIG. 2). As most effective provedwith 81.5% the Ac-heparin (SH4).

[0137] If the effects of the Ac-heparin-coated stents on the plateletloss are considered, then good congruent values result. The correlationof the platelet loss in the perfusate resp. the adhesion of theplatelets to the offered surfaces show the reliability of themeasurements.

[0138] 3.4.1 Covalent Hemocompatible Coating of Stents

[0139] Not expanded stents of medicinal stainless steel LVM 316 weredegreased in the ultrasonic bath for 15 minutes with acetone and ethanoland dried at 100° C. in the drying closet. Then they were dipped for 5minutes into a 2% solution of 3-aminopropyltriethoxysilane in a mixtureof ethanol/water (50/50: (v/v)) and then dried for 5 minutes at 100° C.Afterwards the stents were washed with demineralised water over night.

[0140] 3 mg desulphated and reacetylated heparin were dissolved at 4° C.in 30 ml 0.1 M MES-buffer (2-(N-morpholino)ethanesulphonic acid) pH 4.75and mixed with 30 mgN-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide-methyl-p-toluenesulphonate.In this solution 10 stents were stirred for 15 hours at 4° C. Then theywere rinsed with water, 4 M NaCl solution and water in each case for 2hours.

[0141] 3.4.2 Determination of the Glucosamine Content of the CoatedStents by HPLC

[0142] Hydrolysis: the coated stents are given in small hydrolysis tubesand are abandoned with 3 ml 3 M HCl for exactly one minute at roomtemperature. The metal probes are removed and the tubes are incubatedafter sealing for 16 hours in the drying closet at 100° C. Then they areallowed to cool down, evaporated three times until dryness and taken upin 1 ml de-gased and filtered water and measured contra an alsohydrolysated standard in the HPLC: desulphat. + desulphat. +desulphat. + sample reacet. heparin area reacet. heparin reacet. heparinstent area [g/sample] [cm²] [g/cm²] [pmol/cm²] 1 129.021 2.70647E − 070.74 3.65739E − 07 41.92 2 125.615 2.63502E − 07 0.74 3.56084E − 0740.82 3  98.244 1.93072E − 07 0.74 2.60908E − 07 29.91 4 105.4552.07243E − 07 0.74 2.80058E − 07 32.10 5 119.061 2.33982E − 07 0.743.16192E − 07 36.24 6 129.202 2.53911E − 07 0.74 3.43124E − 07 39.33 7125.766 2.53957E − 07 0.74 3.43185E − 07 39.34

Example 4

[0143] In Vivo Examination of Coated Coronary Stents (FIG. 5)

[0144] 4.1. In Vivo Examinations of Coronary Stents Coated withAc-Heparin

[0145] Due to the data on hemocompatibility, which Ac-heparin yielded inthe in-vitro experiments, the suitability of the Ac-heparin surface asathrombogeneous coating of metal stents was discussed in vivo (animalexperiment).

[0146] The target of the experiments was primarily to evaluate theinfluence of the Ac-heparin coating on the stent induced vesselreaction. Besides the registration of possible thrombotic events therelevant parameters for restenotic processes like neointima area, vessellumen and stenosis degree were recorded. For the examinations 6-9 monthold domestic porks were used, one for the validation of stents for along time established and approved animal model.

[0147] As expected in these experiments neither acute, subacute nor lateacute thrombotic events were registered, what may be assessed as prooffor the athrombogeneous properties of Ac-heparin.

[0148] After four weeks the animals were dispatched (euthanized), thestented coronary artery segments extracted and histomorphometricallyanalysed. Indications to a possible acute or subchronic toxicity,allergisation reactions or ulterior irritations as consequence of theimplantation of Ac-heparin coated stents are not observed during thecomplete experimental phase, especially in the histologic examination.During the stent implantation as well as the follow-upcoronary-angiographic data sets were ascertained, which permit aninterpretation with regard to the vessel reaction to the stentimplantation.

[0149] The difference between the uncoated control stent and theAc-heparin coated stent is unambiguous. The generation of a distinctneointima layer is in case of the uncoated control stent very wellobservable. Already after four weeks the proliferation promotionaleffect of the uncoated stent surface on the surrounding tissue occurs insuch a degree, that ultimately the danger of the vessel occlusion in thestent area is given.

[0150] Contrary in case of the Ac-heparin coated stents a clearlythinner neointima layer is observed, which argues for a well modulatedingrowth of the stent under maintenance of a wide, free vessel lumen.

[0151] The detailed histomorphometric and coronary angiographic datasubstantiate this conclusion, as it can be observed congruently, thatvia the Ac-heparin coating (SH4) the neointima hyperplasia(“restenosis”) was repressed by about 17-20% in comparison to theuncoated control stent. This result is unexpected and remarkably at thesame time. Surely it is not demanded of an athrombogeneous surface tohave an influence also on processes that lead to a neointimahyperplasia, i.e. to prevent restenoses, in addition to the prepositionof hemocompatible characteristics.

[0152] On the one hand with a dense, permanent occupancy of the stentsurface with Ac-heparin a direct cell contact to the metal surface isprevented. As in technical literature the emission of certain metal ionsinto the implant close tissue is discussed as one probable reason ofrestenosis, an anti-restenoic potency could be founded by one of thecoating caused prevention of a direct metal contact.

[0153] On the other hand such a positive side effect is plausible,because on a passive, athrombogenenous stent surface with the absence ofa platelet aggregation also the proliferative effects of the therebyreleased growth factors are to be missed. Thus an important, startingfrom the lumen side, stimulus of the neointimal proliferation isomitted.

Example 5

[0154] Coating of the Stents with Taxol by the Spraying Method

[0155] The via example 1 and example 2 prepared not expanded stents arebalanced and horizontally hung onto a thin metal bar (d=0.2 mm), whichis stuck on the rotation axis of the rotation and feed equipment androtates with 28 r/min. The stents are fixed in that way, that the insideof the stents does not touch the bar. At a feeding amplitude of 2.2 cmand a feeding velocity of 4 cm/s and a distance of 6 cm between stentand spray valve the stent is sprayed with the particular spray solution.After the drying (about 15 minutes) at room temperature and proximate inthe fume hood over night it is balanced again.

[0156] Fabrication of the spray solution: 44 mg taxol are dissolved in 6g chloroform. stent no. before coating after coating coating mass 10.0194 g 0.0197 g 0.30 m

Example 6

[0157] Determination of the Elution Behaviour in PBS-Buffer

[0158] Per stent in a sufficient small flask 2 ml PBS-buffer is added,sealed with para-film and incubated in the drying closet at 37° C. Afterexpiry of the chosen time intervals in each case the excess solution isdepipetted and its UV absorption at 306 nm is measured.

1. Medical device, wherein at least one part of the surface of themedical device is coated directly or via at least one interjacentbiostable and/or biodegradable layer with a hemocompatible layercomprising at least one compound of the formula 1

wherein n represents an integer between 4 and 1050, Y represents aresidue —CHO, —COCH₃, —COC₂H₅, —COC₃H₇, —COC₄H₉, —COC₅H₁₁, —COCH(CH₃)₂,—COCH₂CH(CH₃)₂; —COC H(CH₃)C₂H₅, —COC(CH₃)₃, —CH₂COO—, —C₂H₄COO^(−, —C)₃H₆COO—, —C₄H₈COO—, as well as salts of these compounds, and on, inand/or under the hemocompatible layer the active agent paclitaxel ispresent.
 2. Medical device according to claim 1, wherein Y representsthe residue —CHO, —COCH₃, —COC₂H₅, —COC₃H₇, as well as salts of thesecompounds.
 3. Medical device according to claim 2, wherein Y is —COCH₃.4. Medical device according to claim 1, wherein the hemocompatible layeris directly placed on the surface of the medical device and onto saidhemocompatible layer paclitaxel as well as mixtures of these activeagents are deposited.
 5. Medical device according to claim 1, whereinunder the hemocompatible layer or between two hemocompatible layers atleast one biostable and/or biodegradable layer is present.
 6. Medicaldevice according to claim 1, wherein the hemocompatible layer is coatedcompletely or/and incompletely with at least one additional, above lyingbiostable and/or biodegradable layer.
 7. Medical device according toclaim 1, in which at least one active agent layer of paclitaxel ispresent between the biostable and the hemocompatible layer.
 8. Medicaldevice according to claim 1, in which paclitaxel is bound covalentlyand/or adhesively in and/or on the hemocompatible layer and/or thebiostable and/or the biodegradable layer.
 9. Medical device according toclaim 1, characterised in, that as biodegradable substances for thebiodegradable layer polyvalerolactones, poly-ε-decalactones,polylactonic acid, polyglycolic acid, polylactides, polyglycolides,copolymers of the polylactides and polyglycolides, poly-ε-caprolactone,polyhydroxybutanoic acid, polyhydroxybutyrates, polyhydroxyvalerates,polyhydroxybutyrate-co-valerates, poly(1,4-dioxane-2,3-diones),poly(1,3-dioxane-2-one), poly-para-dioxanones, polyanhydrides aspolymaleic anhydrides, polyhydroxymethacrylates, fibrin,polycyanoacrylates, polycaprolactonedimethylacrylates, poly-b-maleicacid, polycaprolactonebutyl-acrylates, multiblock polymers as e.g. fromoligocaprolactonedioles and oligodioxanonedioles, polyetherestermultiblock polymers as e.g. PEG and poly(butyleneterephtalates),polypivotolactones, polyglycolic acid trimethyl-carbonates,polycaprolactone-glycolides, poly(g-ethylglutamate),poly(DTH-iminocarbonate), poly(DTE-co-DT-carbonate),poly(bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic acidtrimethyl-carbonates, polytrimethylcarbonates, polyiminocarbonates,poly(N-vinyl)-pyrrolidone, polyvinylalcoholes, polyesteramides,glycolated polyesters, polyphosphoesters, polyphosphazenes,poly[p-carboxyphenoxy)propane], polyhydroxypentane acid, polyanhydrides,polyethyleneoxide-propyleneoxide, soft polyurethanes, polyurethanes withamino acid rests in the backbone, polyetheresters as polyethyleneoxide,polyalkeneoxalates, polyorthoesters as well as their copolymers, lipids,carrageenans, fibrinogen, starch, collagen, protein based polymers,polyamino acids, synthetic polyamino acids, zein, modified zein,polyhydroxyalkanoates, pectic acid, actinic acid, modified and nonmodified fibrin and casein, carboxymethylsulphate, albumin, moreoverhyaluronic acid, chitosane and its derivatives, heparansulphates and itsderivatives, heparin, chondroitinsulphate, dextran, b-cyclodextrins,copolymers with PEG and polypropyleneglycol, gummi arabicum, guar,gelatine, collagen, collagen-N-Hydroxysuccinimide, lipids,phospholipids, modifications and copolymers and/or mixtures of aforementioned substances are used.
 10. Medical device according to claim 1,characterised in, that as biostable substances for the biostable layerpolyacrylic acid and polyacrylates as polymethylmethacrylate,polybutylmethacrylate, polyacrylamide, polyacrylonitriles, polyamides,polyetheramides, polyethylenamine, polyimides, polycarbonates,polycarbourethanes, polyvinylketones, polyvinylhalogenides,polyvinylidenhalogenides, polyvinylethers, polyisobutylenes,polyvinylaromates, polyvinylesters, polyvinylpyrollidones,polyoxymethylenes, polytetramethyleneoxide, polyethylene, polypropylene,polytetrafluoroethylene, polyurethanes, polyetherurethanes,silicone-polyetherurethanes, silicone-polyurethanes,silicone-polycarbonate-urethanes, polyolefine elastomeres,polyisobutylenes, EPDM gums, fluorosilicones, carboxymethylchitosanes,polyaryletheretherketones, polyetheretherketones,polyethylenterephthalate, polyvalerates, carboxymethylcellulose,cellulose, rayon, rayontriacetates, cellulosenitrates,celluloseacetates, hydroxyethylcellulose, cellulosebutyrates,celluloseacetatebutyrates, ethylvinylacetate copolymers, polysulphones,epoxy resins, ABS resins, EPDM gums, silicones as polysiloxanes,polydimethylsiloxanes, polyvinylhalogenes and copolymers,celluloseethers, cellulosetriacetates, chitosanes and copolymers and/ormixtures of these substances are used.
 11. Medical device according toclaim 1, whereas instead of the active agent paclitaxel one of thefollowing active agents is used: simvastatin,2-methylthiazolidine-2,4-dicarboxylic acid and the correspondent sodiumsalt, macrocyclic suboxide (MCS), derivatives of MCS, activated proteinC (aPC), PETN, trapidil, β-estradiol as well as mixtures of these activeagents or mixtures of one of these active agents with paclitaxel. 12.Medical device according to claim 1, characterised in, that the medicaldevice comprises prostheses, organs, vessels, aortas, heart valves,tubes, organ spareparts, implants, fibers, hollow fibers, stents, hollowneedles, syringes, membranes, tinned goods, blood containers,titrimetric plates, pacemakers, adsorbing media, chromatography media,chromatography columns, dialyzers, connexion parts, sensors, valves,centrifugal chambers, recuperators, endoscopes, filters, pump chambers.13. Medical device according to claim 12, characterised in, that themedical device is a stent.
 14. Stents according to claim 13, wherein thepolymer is deposited in amounts between 0.01 mg to 3 mg/layer, preferredbetween 0.20 mg to 1 mg and especially preferred between 0.2 mg to 0.5mg/layer.
 15. Stent according to claim 13, characterised in, that theactive agent is used in a pharmaceutically active concentration of0.001-10 mg per cm² stent surface and per layer.
 16. Use of the stentaccording to claim 13 for the prevention or reduction of restenosis. 17.Use of the stent according to claim 13 for continuous release ofpaclitaxel, simvastatin, 2-methylthiazolidine-2,4-dicarboxylic sodiumsalt, macrocyclic suboxide (MCS), derivatives of MCS, activated proteinC (aPC), PETN, trapidil and/or β-estradiol.
 18. Use of the medicaldevice according to claim 1 for the direct contact with blood.
 19. Useof the medical device according to claim 1 for prevention or reductionof the unspecific adhesion and/or deposition of proteins on the coatedsurfaces of the medical devices.
 20. Use according to claim 18,characterised in, that the hemocompatibly coated surface of the medicaldevice is a surface of micro-titer plates or other carrier media fordetection processes.
 21. Use according to claim 18, characterised in,that the hemocompatibly coated surface of the medical device is thesurface of adsorber media or chromatography media.
 22. Method for thehemocompatible coating of biological and/or artificial surfaces ofmedical devices comprising the following steps: a) providing a surfaceof a medical device and b) deposition of at least one compound of thegeneral formula 1 according to claim 1 as hemocompatible layer on thissurface and/or b′) deposition of a biostable and/or biodegradable layeron the surface of the medical device or the hemocompatible layer. 23.Method according to claim 22, wherein the hemocompatible layer or thebiostable and/or biodegradable layer is coated via dipping or sprayingmethod with at least one biodegradable and/or biostabile layer whichconspires paclitaxel covalently and/or adhesively bound.
 24. Methodaccording to claim 22 comprising the further step c): c. deposition ofpaclitaxel in and/or on the hemocompatible layer or the biostable and/orbiodegradable layer.
 25. Method according to claim 24, whereinpaclitaxel is implemented and/or deposited via dipping or sprayingmethods on and/or in the hemocompatible layer or the biostable and/orbiodegredable layer and/or is bound via covalent and/or adhesivecoupling to the hemocompatible layer or the biostable and/orbiodegradable layer.
 26. Method according to claim 22, comprising thefurther step d) or d′): d. deposition of at least one biodegradablelayer and/or at least one biostable and/or biodegradable layer on thehemocompatible layer or the layer of paclitaxel respectively, or d′)deposition of at least one compound of the general formula 1 accordingto claim 1 as hemocompatible layer on the biostable and/or biodegradablelayer or the layer of paclitaxel.
 27. Method according to claim 22,comprising the further step e.): e. deposition of paclitaxel in and/oron the at least one biodegradable and/or biostable layer or thehemocompatible layer.
 28. Method according to claim 27, whereinpaclitaxel is deposited and/or implemented via dipping or sprayingmethods on and/or in the at least one biodegradable and/or biostablelayer or the hemocompatible layer and/or is bound via covalent and/oradhesive coupling to the at least one biodegradable and/or biostablelayer or the hemocompatible layer.
 29. Method according to claim 22,wherein the biostable and/or biodegradable layer is covalently and/oradhesively bound on the surface of the medical device and thehemocompatible layer is covalently bound to the biostable and/orbiodegradable layer and covers it completely or incompletely.
 30. Methodaccording to claim 22, characterised in, that the hemocompatible layercomprises heparin of native origin of regioselectively synthesisedderivatives of different sulphation coefficients and acylationcoefficients in the molecular weight range of the pentasaccharide, whichis responsible for the antithrombotic activity, up to the standardmolecular weight of the purchasable heparin of 13 kD, heparansulphateand its derivatives, oligo- and polysaccharides of the erythrocyticglycocalix, desulphated and N-reacetylated heparin, N-carboxymethylatedand/or partially N-acetylated chitosan as well as mixtures of thesesubstances.
 31. Method according to claim 22, characterised in, that asbiodegradable substances for the biodegradable layer polyvalerolactones,poly-e-decalactones, polylactonic acid, polyglycolic acid, polylactides,polyglycolides, copolymers of the polylactides and polyglycolides,poly-ε-caprolactone, polyhydroxybutanoic acid, polyhydroxybutyrates,polyhydroxyvalerates, polyhydroxybutyrate-co-valerates,poly(1,4-dioxane-2,3-diones), poly(1,3-dioxane-2-one), poly-para-dioxanones, polyanhydrides as polymaleic anhyd rides,polyhydroxymethacrylates, fibrin, polycyanoacrylates,polycaprolactonedimethylacrylates, poly-b-maleic acid,polycaprolactonebutyl-acrylates, multiblock polymers as e.g. fromoligocaprolactonedioles and oligodioxanonedioles, polyetherestermultiblock polymers as .e.g. PEG and poly(butyleneterephtalates),polypivotolactones, polyglycolic acid trimethyl-carbonates,polycaprolactone-glycolides, poly(g-ethylglutamate),poly(DTH-iminocarbonate), poly(DTE-co-DT-carbonate),poly(bisphenol-A-iminocarbonate), polyorthoesters, polyglycolic acidtrimethyl-carbonates, polytrimethylcarbonates, polyiminocarbonates,poly(N-vinyl)-pyrrolidone, polyvinylalcoholes, polyesteramides,glycolated polyesters, polyphosphoesters, polyphosphazenes,poly[p-carboxyphenoxy)propane], polyhydroxypentane acid, polyanhydrides,polyethyleneoxide-propyleneoxide, soft polyurethanes, polyurethanes withamino acid rests in the backbone, polyetheresters as polyethyleneoxide,polyalkeneoxalates, polyorthoesters as well as their copolymers,lipides, carrageenanes, fibrinogen, starch, collagen, protein basedpolymers, polyamino acids, synthetic polyamino acids, zein, modifiedzein, polyhydroxyalkanoates, pectic acid, actinic acid, modified and nonmodified fibrin and casein, carboxymethylsulphate, albumin, moreoverhyaluronic acid, chitosane and its derivatives, heparansulphates and itsderivatives, heparin, chondroitinsulphate, dextran, β-cyclodextrins,copolymers with PEG and polypropyleneglycol, gummi arabicum, guar,gelatine, collagen, collagen-N-Hydroxysuccinimide, lipids,phospholipids, modifications and copolymers and/or mixtures of aforementioned substances are used.
 32. Method according to claim 22,characterised in, that as biostable substances for the biostable layerpolyacrylic acid and polyacrylates as polymethylmethacrylate,polybutylmethacrylate, polyacrylamide, polyacrylonitriles, polyamides,polyetheramides, polyethylenamine, polyimides, polycarbonates,polycarbourethanes, polyvinylketones, polyvinylhalogenides,polyvinylidenhalogenides, polyvinylethers, polyisobutylenes,polyvinylaromates, polyvinylesters, polyvinylpyrollidones,polyoxymethylenes, polytetramethyleneoxide, polyethylene, polypropylene,polytetrafluoroethylene, polyurethanes, polyetherurethanes,silicone-polyetherurethanes, silicone-polyurethanes,silicone-polycarbonate-urethanes, polyolefine elastomeres,polyisobutylenes, EPDM gums, fluorosilicones, carboxymethylchitosanes,polyaryletheretherketones, polyetheretherketones,polyethylenterephthalate, polyvalerates, carboxymethylcellulose,cellulose, rayon, rayontriacetates, cellulosenitrates,celluloseacetates, hydroxyethylcellulose, cellulosebutyrates,celluloseacetatebutyrates, ethylvinylacetate copolymers, polysulphones,epoxy resins, ABS resins, EPDM gums, silicones as polysiloxanes,polydimethylsiloxanes, polyvinylhalogenes and copolymers,celluloseethers, cellulosetriacetates, chitosanes and copolymers and/ormixtures of these substances are used.
 33. Method according to claim 22,characterised in, that the deposition of the polysaccharides of theformula 1 according to claim 1 is achieved via hydrophobic interactions,van der Waals forces, electrostatic interactions, hydrogen bonds, ionicinteractions, cross-linking and/or covalent bonding.
 34. Methodaccording to claim 22, wherein instead of the active agent paclitaxelone of the following active agents is used: simvastatin,2-methylthiazolidine-2,4-dicarboxylic sodium salt, macrocyclic suboxide(MCS), derivatives of MCS, activated protein C (aPC), PETN, trapidil,β-estradiol as well as mixtures of these active agents or mixtures ofone of these active agents with paclitaxel.
 35. Medical device availableby the method according to claim 22.